Molecular Means for 3' Splice Site Recognition by U2AF35

U2AF35 识别 3 剪接位点的分子方法

• 批准号: 8324344
• 负责人: Kholiswa M Laird
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8324344
• 关键词: 3' Splice Site; Address; Adopted; Affinity; Alternative Splicing; Binding; Calorimetry; Cell physiology; Code; Commit; Complex; Consensus; Consensus Sequence; Crystallization; Cysteine; Data; Diagnosis; Dinucleoside Phosphates; Disease; Dissociation; Electrophoretic Mobility Shift Assay; Equilibrium; Event; Fluorescein; Fluorescence Anisotropy; Gene Expression Regulation; Goals; Hereditary Disease; Histidine; Human; Human Genetics; Kinetics; Knowledge; Label; Length; Link; Literature; Malignant Neoplasms; Methods; Molecular; Mutate; Neuromuscular Diseases; Oligonucleotides; Organism; Outcome; Point Mutation; Positioning Attribute; Production; Property; Protein Binding; Proteins; RNA; RNA Binding; RNA Sequences; RNA Splice Sites; RNA Splicing; Recruitment Activity; Regulation; Research; Resolution; Roentgen Rays; Role; Sequence Homology; Site; Site-Directed Mutagenesis; Solutions; Source; Specific qualifier value; Specificity; Spliceosome Assembly Pathway; Spliceosomes; Staging; Structure; Surface Plasmon Resonance; Techniques; Therapeutic; Thermodynamics; Titrations; Transcript; Variant; Work; X-Ray Crystallography; Zinc; base; design; leukemia; mRNA Precursor; mutant; research study

DESCRIPTION (provided by applicant): The splicing of pre-mRNA transcript coding regions leads to the production of a variety of key protein variants essential to many cellular processes in humans and complex organisms. To date, it is still unknown as to how the splicing machinery termed as the "spliceosome" correctly recognizes pre-mRNA splice sites. During the early stages of spliceosome assembly, an essential auxiliary protein factor, U2AF, binds to a polypyrimidine and 'AG' dinucleotide consensus sequence at the 3' splice site. This is a crucial step in splice site assembly as it commits pre-mRNA substrates to be spliced. U2AF is a heterodimer comprised of U2AF65 and U2AF35. The role of U2AF65 at the 3' splice site is to bind the polypyrimidine tract and recruit other splicing factors to the pre-mRNA where its interactions have been well characterized. Currently, it is known that U2AF35 contacts the 'AG' consensus site, however, the overall contributions of U2AF35 at the 3' splice site are not well known. The aims of this proposed research intend to address the following critical questions, still un-answered in the pre-mRNA splicing field: (i) what is the fundamental means of U2AF35 contacting the pre-mRNA at the correct 3' splice site? and (ii) what is the structural basis for this interaction? The significance of these questions relies upon the fact that splice site recognition by U2AF is a critical step in gene regulation, where aberrant regulation of splicing has been linked to various diseases in humans. Furthermore, the domains of U2AF35 that contact the 3' splice site 'AG' consensus sequence and the spatial arrangement of these interactions is currently unknown. In this proposed research we will use a variety of biophysical techniques to characterize U2AF35 in complex with RNA substrates including the 3' splice site 'AG' consensus sequence. Briefly, we will utilize fluorescence anisotropy, electrophoretic mobility shifts assays (EMSA) and site-directed mutagenesis to determine the affinity and specificity of U2AF35/RNA interactions. Surface Plasmon resonance (SPR) and isothermal titration calorimetry (ITC) methods will be used to provide a kinetic and thermodynamic description of these interactions. Using X-ray crystallography we will determine the structure of a U2AF35 variant bound to an oligonucleotide containing the 3' splice site 'AG' consensus sequence. Finally, we will collect small angle X- ray scattering (SAXS) data on full-length U2AF both in the presence and absence of RNA to provide the molecular envelop of U2AF/RNA interactions in solution.

描述（由申请人提供）：前MRNA转录物编码区域的剪接导致产生各种关键蛋白质变异，这对于人类和复杂生物的许多细胞过程必不可少。迄今为止，对于如何正确识别前MRNA剪接位点的剪接机械如何称为“剪接”仍然尚不清楚。在剪接体组装的早期阶段，必需的辅助蛋白因子U2AF与3'剪接位点上的多嘧啶和“ Ag”二核苷酸共识序列结合。这是剪接位点组件中的关键步骤，因为它提交了要剪接的前MRNA底物。 U2AF是由U2AF65和U2AF35组成的异二聚体。 U2AF65在3'剪接位点的作用是结合息肉嘧啶界，并将其他剪接因子募集到其相互作用的前MRNA中。目前，众所周知，U2AF35与“ AG”共识站点接触，但是，U2AF35在3'剪接站点的总体贡献并不众所周知。 这项拟议的研究的目的是解决以下关键问题，但仍未在MRNA剪接领域中进行交流：（i）U2AF35在正确的3'剪接网站上与前MRNA联系的基本手段是什么？ （ii）这种相互作用的结构基础是什么？这些问题的意义取决于以下事实：U2AF的剪接部位识别是基因调节的关键一步，在基因调节中，剪接的异常调节与人类的各种疾病有关。此外，目前尚不清楚与3“剪接位点”共识序列和这些相互作用的空间排列接触的U2AF35的域。 在这项拟议的研究中，我们将使用各种生物物理技术来表征与RNA底物在复合物中的U2AF35，包括3个“剪接位点” AG”共识序列。简而言之，我们将利用荧光各向异性，电泳迁移率转移测定（EMSA）和定向诱变来确定U2AF35/RNA相互作用的亲和力和特异性。表面等离子体共振（SPR）和等温滴定量热法（ITC）方法将用于提供这些相互作用的动力学和热力学描述。使用X射线晶体学，我们将确定与包含3个“剪接位点“ Ag”共识序列的寡核苷酸结合的U2AF35变体的结构。最后，在存在和不存在RNA的情况下，我们将在全长U2AF上收集小角度X射线散射（SAXS）数据，以提供溶液中U2AF/RNA相互作用的分子包络。